Congenital heart defects (CHDs) are the leading non-infectious cause of death in the first year of life; however, the etiology of most lesions is unknown. Hairy-related transcription factors (Hrt1, Hrt2 and Hrt3) are transcriptional repressors that are expressed in the somites, cardiac outflow tract, pulmonary arteries, and have mutually exclusive expression domains in the atria and ventricles. We found that Hrt2 forms a complex with Gata4, a transcription factor necessary for normal human cardiac development, that results in repression of Gata4 transactivation. Hrt2 repression of Gata4 was disrupted by the kinase Akt1. We have also identified mutations in the coding regions of Hrt2 in patients with congenital heart disease that suggest a novel Hrt domain necessary for repression. We hypothesize that (1) Hrt2's negative regulation of Gata4-dependent transactivation is sensitive to a growth signal, and (2) that mutations in Hrt2 found in patients with congenital heart malformations reveal a novel and critical domain for Hrt function during cardiogenesis. The specific aims to be addressed in this proposal include: Specific Aim 1: To elucidate the biologic significance of the Hrt2-Gata4 complex and the mechanism(s) by which Hrt2-mediated repression of GATA-dependent cardiac gene expression is sensitive to Akt1. We will study the functional consequences of Hrt2 on chromatin acetylation of endogenous GATA-dependent cardiac genes and examine the role of the orange domain in mediating Akt1-responsive protein-protein interactions. Specific Aim 2: To determine the mechanisms by which clustered human HRT2 mutations found in CHDs affect Hrt2 function. We will conduct detailed in vitro assays to identify co-factors dependent on this novel domain, and we will generate mice harboring these mutations to study their functional consequences in vivo.